Complete genomic sequence of rabies virus from an ethiopian wolf.

Ethiopian wolves are the rarest canid in the world, with only 500 found in the Ethiopian highlands. Rabies poses the most immediate threat to their survival, causing epizootic cycles of mass mortality. The complete genome sequence of a rabies virus (RABV) derived from an Ethiopian wolf during the most recent epizootic is reported here.

GENOME ANNOUNCEMENT

Rabies virus (RABV) is the type species of the Lyssavirus genus and the causative agent of rabies. Throughout Africa, domestic dogs are the principal reservoir for RABV (1), transmitting the virus to a wide range of terrestrial mammals, including wildlife, such as the Ethiopian wolf (Canis simensis) (2, 3). The dominant RABV lineage circulating within domestic dogs in Ethiopia is Africa 1a, which is part of the cosmopolitan lineage (4). Long-term population monitoring of Ethiopian wolves, in combination with laboratory testing of all wolf carcasses recovered, indicates that rabies is not endemic in the wolf population; rather, rabies epizootics occur in locations with a high density of wolves (5). Previous outbreaks of rabies in Ethiopian wolves have been effectively controlled with parenteral vaccination (6). A comparison of the complete virus genomes can enable fine mapping of disease transmission events, such as those that trigger Ethiopian wolf RABV epizootics. However, publically available complete genomes for African RABVs are limited. Of the 6 Africa 1 clade genomes available, only one is an Africa-1a bovine isolate from Morocco (accession no. KF155001). Here, we describe full-genome sequencing of an Africa-1a RABV obtained from an infected Ethiopian wolf from the Sanetti Plateau, Bale Mountains, southeastern Ethiopia (RV2985), in August 2014.

RNA from brain material stored in glycerol was prepared for next-generation sequencing on the MiSeq platform. Briefly, TRIzol-extracted viral RNA was depleted of host genomic DNA and rRNA, as described previously (7). Double-stranded (ds)-cDNA was synthesized from 50 ng of RNA using a random cDNA synthesis system (Roche), according to the manufacturer’s instructions. The ds-cDNA was purified using AMPure XP magnetic beads (Beckman Coulter), and 1 ng was used for the Nextera XT DNA sample preparation kit (Illumina). A sequencing library was prepared according to the manufacturer’s instructions and sequenced on an Illumina MiSeq with 2 × 150-bp paired-end reads, according to standard Illumina protocols. The total reads (5,333,189) were mapped to a reference sequence (accession no. KF15501) using the Burrows-Wheeler Aligner (BWA) (version 0.7.5a-r405) (8) and were visualized in Tablet (9). A modified SAMtools/vcfutils (10) script was used to generate an intermediate consensus sequence in which any indels relative to the original reference sequence were appropriately called. The intermediate consensus was used as the reference for subsequent iteration of mapping and consensus calling. The total number of assembled viral reads was 4,750 (0.09% of the total reads). Despite the low proportion of viral sequence detected within the total data set, coverage of the entire genome was obtained (average read depth, 43.7×).

The genetic organization of the Ethiopian wolf RABV genome was consistent with that of other RABV genomes, with a complete genome size of 11,926 nucleotides. The lengths of the individual coding regions were conserved; however, indels were observed in the G-L intergenic region. The Ethiopian wolf RABV genome has the closest homology to the Moroccan RABV genome sequence (accession no. KF155001, 95%), indicating that it belongs to the Africa-1a lineage. Future analysis with domestic dog RABV genomes from Ethiopia will further our understanding of the cross-species transmissions that lead to epizootic cycles observed in the Ethiopian wolf population.

Nucleotide sequence accession number.

The complete genomic sequence of RV2985 has been deposited in GenBank under the accession no. KP723638.